Evidence has been previously presented that the hepatotoxicity of phenacetin is mediated through the deethylated metabolite acetaminophen followed by activation of acetaminophen to a reactive metabolite. N-acetylimidoquinone was postulated to be the reactive metabolite and was believed to be formed by N-hydroxylation of acetaminophen followed by a spontaneous dehydration. Since the half life of N-hydroxyacetaminophen in aqueous solutions at pH 7.4 was recently shown to be 15 minutes, we examined the possibility that N-hydroxyacetaminophen was a metabolite of acetaminophen and N-hydroxyphenacetin. N-Hydroxyacetaminophen was determined to be a major metabolite of N-hydroxyphenacetin by gas chromatography-mass spectrometry and high pressure liquid chromatography but was apparently not a metabolite of acetaminophen. More covalent binding, however, occurred with acetaminophen as a substrate than with N-hydroxyphenacetin. These data suggest that the chemically reactive metabolites of acetaminophen and phenacetin are not mediated by N-hydroxylation of acetaminophen as previously postulated.